Made available in DSpace on 2016-08-10T10:39:18Z (GMT). No. of bitstreams: 1 Hilton Rinaldo Salles Piccelli.pdf: 1013428 bytes, checksum: 900904fd782b76c7a9b8862f4b21bc9e (MD5) Previous issue date: 2008-05-19 / Actinic cheilits (AC) is a premalignant lesion of the lip. In general, patients with actinic cheilitis present with no symptoms and clinical signs do not reflect the hystopathological severity of the lesion, allowing its evolution to invasive cancer. Prognostic indicators for evolution of such lesions have been investigated by different groups, including the detection of alterations in the TP53 gene. A series of 48 patients with actinic cheilitis is evaluated in this study. Clinical signs, histopathological features and imunohistochemistry detection of the p53 protein were evaluated in this group. All the patients were caucasian and 44% had a previous history of non-melanoma skin cancer in another site of the body. The most frequent clinical signs observed in the patients included loss of lip vermilion (75%), pelling of the lip (71%) and atrophy (67%). Eighty-five per cent of the patients did not present, on the first consultation, specific complaints related to actinic cheilitis. Histopathological analysis described dyskeratosis in 95% of the samples, epithelial hyperplasia in 85% and dysplasia in 57% of the cases. munodetection of p53 on the suprabasal layer was diffuse in 8 patients. Among hem, 12% of the samples showed dyskeratosis, 87% hyperplasia and 100% presented epithelium dysplasia. The diffuse imunodetection of the p53 protein on he suprabasal layer correlated positively with the presence of epithelial dysplasia (p many 0,005). According to different reports, dysplasia is the most significant prognostic factor to the evolution of actinic cheilitis to squamous cell carcinoma of he lip. We here suggest that the imunodetection of p53 on the suprabasal layer of actinic cheilitis samples might also represent an important prognostic factor to such premalignant lesions. / A queilite actinica (QA) é uma lesão pré-maligna do lábio. Em geral, os pacientes são assintomáticos e os sinais clínicos não refletem a gravidade histopatológica da lesão, permitindo sua evolução para o câncer invasor. Marcadores prognósticos para esta evolução têm sido investigados, incluindo a detecção de alterações no gene TP53. Uma série de 48 pacientes com QA é avaliada neste estudo. Sinais clínicos, aspectos histopatológicos e a imunodetecção da proteína p53 foram avaliados no grupo. Todos os pacientes eram brancos e 44% apresentaram história prévia de câncer de pele tipo não- melanoma em outro sítio corporal. Os sinais clínicos mais freqüentes incluíram perda do vermelhão do lábio (75%), descamação (71%) e atrofia (67%). À primeira consulta, 85% dos pacientes não apresentavam queixas específicas relacionadas à QA. No exame histopatológico, verificou-se disceratose em 95% dos casos, hiperplasia epitelial em 85% e displasia em 57% dos casos. A imunodetecção de p53 foi difusa e mais intensa na camada suprabasal em 8 pacientes. Dentre eles, 12% apresentavam disceratose, 87% hiperplasia e 100% apresentavam displasia do epitélio (p menor que 0,005). A imunodetecção difusa da proteína p53 na camada suprabasal correlacionou-se positivamente com a presença de displasia epitelial. De acordo com a literatura, a displasia representa o fator prognóstico mais significativo para a evolução da QA para carcinoma do lábio. Assim, sugerimos que a imunodetecção de p53 na camada suprabasal represente também um fator prognóstico para a queilite actínica.

by Yuen Wai Fan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 211-232). / Abstracts in English and Chinese. / Acknowledgements --- p.i / List of Publications and Abstracts --- p.ii / Abbreviations --- p.iv / Abstract --- p.xi / Abstract in Chinese --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Contents --- p.xxiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Hyperthermia --- p.2 / Chapter 1.1.1 --- History of Hyperthermia --- p.2 / Chapter 1.1.2 --- Biological Functions of Hyperthermia --- p.3 / Chapter 1.1.3 --- Clinical Application of Hyperthermia --- p.4 / Chapter 1.1.3.1 --- Whole-body Hyperthermia --- p.4 / Chapter 1.1.3.2 --- Regional Hyperthermia --- p.4 / Chapter 1.1.3.3 --- Local Hyperthermia --- p.5 / Chapter 1.1.4 --- Combination Therapy --- p.5 / Chapter 1.1.4.1 --- Combined treatment with Hyperthermia and Radiotherapy --- p.6 / Chapter 1.1.4.2 --- Combined treatment with Hyperthermia and Chemotherapy --- p.6 / Chapter 1.2 --- Tumour Necrosis Factor --- p.9 / Chapter 1.2.1 --- History of Tumour Necrosis Factor --- p.9 / Chapter 1.2.2 --- Sources of TNF-α and TNF-β --- p.9 / Chapter 1.2.3 --- Biological Roles of TNF --- p.10 / Chapter 1.2.3.1 --- Receptors of TNF-α --- p.11 / Chapter 1.2.4 --- Signaling Pathway of TNF --- p.12 / Chapter 1.2.4.1 --- Activation of Death Domain --- p.12 / Chapter 1.2.4.2 --- Activation of Sphingomyelin Pathway --- p.13 / Chapter 1.2.4.3 --- Activation of NF-kB pathway --- p.13 / Chapter 1.3 --- Types of Cell Death: Necrosis and Apoptosis --- p.16 / Chapter 1.3.1 --- Necrosis --- p.16 / Chapter 1.3.2 --- Apoptosis --- p.16 / Chapter 1.4 --- Signaling Pathway in Apoptosis --- p.19 / Chapter 1.4.1 --- Factors Involved in Apoptotic Pathway --- p.19 / Chapter 1.4.1.1 --- Caspases --- p.19 / Chapter 1.4.1.2 --- Death Substrates --- p.20 / Chapter 1.4.1.3 --- Bcl-2 Protein Family --- p.21 / Chapter 1.4.1.4 --- Role of Mitochondria --- p.23 / Chapter 1.5 --- Objectives of the Project --- p.26 / Chapter Chapter 2. --- Materials and Methods --- p.28 / Chapter 2.1 --- Materials --- p.29 / Chapter 2.1.1 --- Culture of Cells --- p.34 / Chapter 2.1.1.1 --- "TNF-α Sensitive Cell Line, L929" --- p.34 / Chapter 2.1.1.2 --- "TNF-α Resistance Cell Line, L929-11E" --- p.34 / Chapter 2.1.1.3 --- Preservation of Cells --- p.35 / Chapter 2.1.2 --- Culture Media --- p.36 / Chapter 2.1.2.1 --- RPMI 1640 (Phenol Red Medium) --- p.36 / Chapter 2.1.2.2 --- RPMI 1640 (Phenol Red-Free Medium) --- p.36 / Chapter 2.1.3 --- Buffers and Reagents --- p.37 / Chapter 2.1.3.1 --- Preparation of Buffers --- p.37 / Chapter 2.1.3.2 --- Buffer for Common Use --- p.37 / Chapter 2.1.3.3 --- Reagents for Annexin-V-FITC/PI assay --- p.37 / Chapter 2.1.3.4 --- Reagents for Cytotoxicity Assay --- p.37 / Chapter 2.1.3.5 --- Reagents for Molecular Biology Work --- p.38 / Chapter 2.1.3.6 --- Reagents for Western Blotting Analysis --- p.38 / Chapter 2.1.4 --- Chemicals --- p.40 / Chapter 2.1.4.1 --- Recombinant Murine TNF-α --- p.40 / Chapter 2.1.4.2 --- Dye for Cytotoxicity Assay --- p.41 / Chapter 2.1.4.3 --- Fluorescence Dyes --- p.41 / Chapter 2.1.4.4 --- Chemicals Related to Mitochondrial Studies --- p.41 / Chapter 2.1.4.5 --- Inhibitors of Caspases --- p.42 / Chapter 2.1.4.6 --- Antibodies for Western Blotting --- p.42 / Chapter 2.1.4.7 --- Other Chemicals --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Treatment with TNF-α --- p.44 / Chapter 2.2.2 --- Treatment with Hyperthermia --- p.44 / Chapter 2.2.3 --- In vitro Cell Cytotoxicity Assay --- p.45 / Chapter 2.2.4 --- Flow Cytometry --- p.46 / Chapter 2.2.4.1 --- Introduction --- p.46 / Chapter 2.2.4.2 --- Analysis by FCM --- p.48 / Chapter 2.2.4.3 --- Determination of Apoptotic and Late Apoptotic/Necrotic Cells with Annexin-V-FITC/PI Cytometric Analysis --- p.50 / Chapter 2.2.4.4 --- Determination of Mitochondrial Membrane Potential (ΔΨm) --- p.51 / Chapter 2.2.4.5 --- Determination of Hydrogen Peroxide (H202) Release --- p.52 / Chapter 2.2.4.6 --- Determination of Intracellular Free Calcium ([Ca2+]i) Level --- p.52 / Chapter 2.2.4.7 --- Determination of the Relationship of ΔΨm and [Ca2+]i Level --- p.53 / Chapter 2.2.5 --- Western Blotting Analysis --- p.53 / Chapter 2.2.5.1 --- Preparation of Proteins from Cells --- p.53 / Chapter 2.2.5.2 --- SDS Polyacrylamide Gel Electophoresis (SDS- PAGE) --- p.56 / Chapter 2.2.5.3 --- Electroblotting of Proteins --- p.57 / Chapter 2.2.5.4 --- Probing Antibodies for Proteins --- p.57 / Chapter 2.2.5.5 --- Enhanced Chemiluminescence (ECL) assay --- p.58 / Chapter 2.2.6 --- Reverse Transcriptase Polymerase Chain Reaction --- p.58 / Chapter 2.2.6.1 --- Extraction of RNA by Trizol Reagent --- p.59 / Chapter 2.2.6.2 --- Determination of the Amount of RNA --- p.60 / Chapter 2.2.6.3 --- Agarose Gel Electrophoresis --- p.60 / Chapter 2.2.6.4 --- Reverse Transcription --- p.63 / Chapter 2.2.6.5 --- Polymerase Chain Reaction (PCR) --- p.63 / Chapter 2.2.6.6 --- Design of Primers for Different Genes --- p.64 / Chapter 2.2.6.7 --- Determination of the Number of Cycles in PCR for Different Genes --- p.67 / Chapter 2.2.7 --- Caspase Fluorescent Assay --- p.67 / Chapter 2.2.7.1 --- Caspase-3 or ´ؤ8 Assay --- p.67 / Chapter Chapter 3. --- Results --- p.59 / Chapter 3.1 --- Studies of the Characteristics of L929 and L929-11E cells --- p.70 / Chapter 3.1.1 --- Determination of the Growth Curve of L929 and L929-11E Cells --- p.70 / Chapter 3.2 --- Studies on the Effect of TNF-α on L929 and L929-11E Cells --- p.73 / Chapter 3.2.1 --- TNF-α Induced Cell Death in L929 Cells but not in L929- 11E Cells --- p.73 / Chapter 3.2.2 --- TNF-α Induced Apoptosis in a Time-dependent Manner in L929Cells but not in L929-11E Cells --- p.80 / Chapter 3.2.3 --- TNF-α Induced Mitochondrial Membrane Depolarization in a Time-dependent Manner in L929 Cells but notin L929-11E Cells --- p.87 / Chapter 3.2.4 --- TNF-α Induced Cytochrome c Release in a Time- dependent Manner in L929 Cells but not in L929-11E Cells --- p.92 / Chapter 3.3 --- Effect of Hyperthermia on L929 and L929-11E Cells --- p.96 / Chapter 3.3.1 --- Introduction --- p.95 / Chapter 3.3.2 --- Hyperthermia Induced Apoptosis in L929 and L929-11E Cells --- p.96 / Chapter 3.3.3 --- Effect of Hyperthermia on Mitochondrial Membrane Depolarization --- p.100 / Chapter 3.3.4 --- Hyperthermia Induced Cyto c Release in a Time-dependent Manner in L929 and L929-11E Cells --- p.105 / Chapter 3.4 --- Relationship of Hyperthermia and TNF-α with PTP in L929 Cells --- p.107 / Chapter 3.5 --- Effect of TNF-α and Hyperthermia on the Level of Hydrogen Peroxide (H202) in L929 and L929-11E Cells --- p.114 / Chapter 3.5.1 --- Introduction --- p.114 / Chapter 3.5.2 --- TNF-α Enhanced the Level of H202 in L929 cells but not in L929-11E Cells --- p.115 / Chapter 3.5.3 --- Hyperthermia Enhanced the Level of H202 in L929 and L929-11E cells --- p.117 / Chapter 3.6 --- Effect of TNF-α and Hyperthermia on the Level of Intracellular Calcium in L929 and L929-11E Cells --- p.122 / Chapter 3.6.1 --- Increase in the Intracellular Calcium Level Induced by TNF-α Was Related to the Mitochondrial Membrane Depolarization in L929 Cells but not in L929-11E Cells --- p.122 / Chapter 3.6.2 --- Hyperthermia Increased the Level of [Ca2+]i in L929 and L929-11E Cells in a Time-dependent Manner --- p.124 / Chapter 3.7 --- Effect of Combined Hyperthermia and TNF-α Treatment on the Induction of Apoptosis in L929 and L929-1 1E Cells --- p.129 / Chapter 3.7.1 --- Combined Treatment with Hyperthermia and TNF- α Induced Apoptosis in Both L929 and L929-11E cells --- p.129 / Chapter 3.7.2 --- Hyperthermia and Its Combined Treatment with TNF-α Induced Mitochondrial Membrane Depolarization in L929 and L929-11E Cells --- p.135 / Chapter 3.8 --- Investigation of the Downstream Apoptotic Pathway in L929 and L929-11E Cells Upon Hyperthermia and TNF-a treatment --- p.142 / Chapter 3.8.1 --- Introduction --- p.142 / Chapter 3.8.2 --- Effect ofTNF-α and Hyperthermia on p53 Expression --- p.142 / Chapter 3.8.3 --- Effect of Hyperthermia and TNF-α on PARP --- p.146 / Chapter 3.8.4 --- Effect of Hyperthermia and TNF-α on Caspase-3 Activity --- p.149 / Chapter 3.8.5 --- Effect of Hyperthermia and TNF-α on Bid protein --- p.158 / Chapter 3.8.6 --- Effect of Hyperthermia and TNF-α on Caspase-8 Activity --- p.165 / Chapter 3.8.7 --- Effect ofTNF-α on TNFR1 Expression --- p.169 / Chapter Chapter 4. --- Discussion / Chapter 4.1 --- TNF-α Induced Apoptosis and Changed the Mitochondrial Activities in L929 Cells --- p.176 / Chapter 4.2 --- L929-11E cells Possessed Resistance Towards TNF-α --- p.187 / Chapter 4.3 --- Hyperthermia Triggered Apoptosis and Changed Mitochondrial Activities in L929 and L929-11E cells --- p.190 / Chapter 4.4 --- Combined hyperthermia and TNF-α treatment induced cell death and changed mitochondria activities in L929 and L929-11E cells --- p.195 / Chapter 4.5 --- Reversal of the TNF-α resistance and Enhancement of Sensitivity Towards Hyperthermia in L929-11E cells --- p.197 / Chapter 4.6 --- Proposed Pathway in the TNF-α- and Hyperthermia-mediated Apoptosis --- p.200 / Chapter 4.7 --- Application of TNF-α and Hyperthermia on Clinical Cancer Treatment --- p.203 / Chapter Chapter 5. --- Future Perspective of the Project --- p.206 / References --- p.210

Les carcinoïdes pulmonaires, les carcinomes neuroendocriniens à grandes cellules (LCNEC) et les mésothéliomes malins sont des tumeurs thoraciques rares, dont l'incidence a augmenté au cours des dernières années. Le diagnostic de ces tumeurs est soumis à la variabilité inter-observateur et les opportunités thérapeutiques sont limitées. De grandes études génomiques visant à les caractériser au niveau moléculaire pourraient aider à mieux comprendre les mécanismes sous-jacents à leur développement et faciliter le diagnostic et le traitement de ces maladies. Mon projet de thèse visait à combler les lacunes dans la compréhension des carcinoïdes pulmonaires, des LCNEC et du mésothéliome péritonéal malin. À la suite des travaux entrepris au cours de ma thèse, nous avons constaté que (1) de la même manière que le mésothéliome pleural, les mésothéliomes péritonéaux sont également caractérisés par des mutations conduisant à la perte d'expression de BAP1, facteur de bon pronostic, (2) les patients atteints d’un LCNEC conservant une expression de RB1 présentent de meilleurs résultats lorsqu’ils sont traité avec une chimiothérapie du cancer du poumon non à petites cellules par rapport à une chimiothérapie du cancer du poumon à petites cellules, (3) les carcinoïdes pulmonaires peuvent être classés en trois groupes moléculaires pertinents sur le plan clinique, et (4) , l'identification de supra carcinoïdes confirme l'existence d'un lien moléculaire entre les néoplasmes neuroendocriniens pulmonaires de faible et de haut grade / Pulmonary carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and malignant mesotheliomas are rare thoracic tumours, which incidence has been increasing over the past years. The diagnosis of these tumours is subjected to inter-observer variability and the therapeutic opportunities are limited. Large genomic studies to characterize them at a molecular level might help to better understand the mechanisms underlying their development, and to help the diagnosis and treatment of these diseases. My thesis project aimed to fill the gap in the understanding of pulmonary carcinoids, LCNEC, and malignant peritoneal mesothelioma. As result of the work undertaken during my thesis, we found that (1) similarly to pleural mesothelioma, peritoneal mesotheliomas are also characterised by mutations leading to the loss of expression of BAP1, which is a factor of good prognostic, (2) LCNEC patients with a remaining expression of RB1 have a better outcome when treated with non-small cell lung cancer chemotherapy in comparison to small-cell lung cancer chemotherapy, (3) pulmonary carcinoids can be classified in three clinically-relevant molecular groups, and (4), the identification of supra carcinoids supports a molecular link between the low and high-grade lung neuroendocrine neoplasms

O exame de tomografia computadorizada é um grande aliado na elaboração do diagnóstico, planejamento do tratamento e proservação de lesões malignas do complexo maxilomandibular. Em muitos casos é considerado o exame de eleição para estes fins. Um dos problemas que ocorrem com a TC, é o aparecimento de artefatos devido a restaurações metálicas, implantes osseointegrados ou dispositivos metálicos de fixação óssea. Estes artefatos atrapalham a interpretação das imagens, dificultando a visualização de lesões. O propósito deste estudo foi avaliar imagens de TC com seções de cortes axiais e coronais e determinar se o tipo de seção pode minimizar o problema dos artefatos metálicos dentários. Para isto, dois avaliadores calibrados analisaram 72 imagens (36 em cortes axiais e 36 em cortes coronais) e graduaram quais imagens eram mais bem interpretadas. Os resultados não mostraram diferenças significantes entre os cortes axiais e coronais para se chegar ao diagnóstico, na presença de artefatos metálicos na imagem, assim como demonstrou boa concordância entre os observadores. Conclui-se que a presença dos artefatos metálicos dentários não foi considerada suficiente para impedir a localização de neoplasias de tecido mole. Não foi detectada uma interferência maior de artefatos dentários metálicos tanto nas imagens axiais como nas coronais com relação à interpretação das lesões. A imagem coronal apresentou uma maior interferência em comparação com a imagem axial. Palavras-chave: tomografia computadorizada, artefatos metálicos, neoplasias malignas dos maxilares. / Computed tomography is an important tool to determinate the diagnosis, treatment and follow up malignant lesions of the maxillomandibular complex. In many cases, it is the election exam to all of these purposes. One problem with CT is the artifacts that appear in the presence of metallic restorations, implants and metallic fixation screws or plates. These artifacts lead to misinterpretations of the images, making difficult to visualize lesions in these areas. The aim of this research was to evaluate CT images with different sections (axial and coronal) e to determine if the type of section can minimize the problem of dental metallic artifacts. Two calibrate examiners (oral and maxillofacial radiologists) analyzed 72 images (36 in axial slices and 36 in coronal slices) and graduate which images were best viewed. The results attested that there were no significant differences between axial and coronal images to reach the diagnosis in the presence of dental metallic artifacts, and there were a good concordance with both examiners. We conclude that the presence of dental metallic artifacts wasn’t sufficient to disturb the precise location neoplasm of the soft tissue. It wasn’t detected any dental metallic interference possible to misdiagnosis neither on axial images nor on coronal images. Coronal images presented a higher interference when comparing with axial images.

Malignant catarrhal fever (MCF) is a lymphoproliferative and inflammatory syndrome affecting primarily ruminant species. The disease, which is often fatal, is most often described as affecting bovids and cervids. No vaccines are available for prevention of MCFV infection. The primary method to control spread of disease is to prevent contact between carriers and clinically susceptible species. There is no known method to control infection of malignant catarrhal fever virus-white-tailed deer variant (MCFV-WTD), as the carrier animal of this virus is unknown. To determine the prevalence of malignant catarrhal fever viruses in Tennessee ruminant populations, blood and/or lymph node samples were collected from farms, animal processing and disposal facilities, and hunter check-in stations from 2006-2008 from several species of animals including cervids, cattle, and goats. Strain-specific real time PCR was developed to detect ovine herpesvirus-2 (OvHV-2), caprine herpesvirus-2 (CpHV-2), and MCFV-WTD DNA. MCFV DNA was detected in all species of ruminants sampled. Although disease related to infection with MCFV-WTD and CpHV-2 has not been reported in Tennessee cattle or cervid populations, MCFV-WTD DNA was detected in 3 percent of cervid samples, and MCFV-WTD and CpHV-2 DNA was detected in 27 and 3 percent respectively of cattle samples from animal disposal facilities that process dead or debilitated animals. One hunter harvested deer (n=781) and 25 cattle (n=165) tested from animal disposal facilities were positive for OvHV-2 DNA. This study demonstrated that healthy cattle and cervids can be infected with OvHV-2 and MCFV-WTD without apparent disease, and dead or debilitated cattle were infected with OvHV-2, MCFV-WTD and CpHV-2 at a higher percentage than healthy herd animals. Prevalence of CpHV-2 in Tennessee goat populations (7%) was significantly lower than reported in other goat populations (73%). Low prevalence of CpHV-2 in Tennessee goat populations likely explains why no evidence of infection was found in cervids tested, and the low prevalence of CpHV-2 infection in dead or debilitated cattle compared to prevalence of infection with OvHV-2 and MCFV-WTD. The discovery of infection in cattle with CpHV-2 and MCFV-WTD opens a new avenue of investigation into the pathology and virulence of MCFV’s in domestic cattle.

Breast Cancer susceptibility gene 1 (BRCA1) & Breast Cancer Breast cancer is one of the most common malignancies affecting women worldwide. About 5-10% of all cases are estimated to be familial. Mutations in the BRCA1 (Breast Cancer susceptibility gene 1) gene account for about 15-20% of inherited breast cancer cases and 60-80% of families predisposed to both breast and ovarian cancer. BRCA1 mutations also result in susceptibility to early-onset breast and ovarian cancer. The human BRCA1 gene encodes a multi-domain 1,863 amino acid nuclear protein that is expressed in a wide variety of adult human tissues. The N-terminal end of BRCA1 contains a RING-finger domain. Exon 11 of BRCA1 contains two nuclear localization signals towards its N-terminal for targeting BRCA1 to the nucleus. The carboxyl terminus contains two BRCT (BRCA1 C-terminal) domains and a transcriptional activation domain. This study was carried out to functionally characterize BRCA1 and to find out the percentage in which BRCA1 gene is mutated in Indian familial breast and/or ovarian cancer families. The work has been divided into three sections: 1. Identification & characterization of a BRCA1 Associated Protein 2 (BAP2). 2. Germ-line BRCA1 mutation Analysis in Indian Breast and/or Ovarian Cancer Families. 3. Characterization of a novel missense mutation (E116K) in BRCA1. BRCA1 is known to interact with large number of proteins and is involved in various cellular functions like tumorigenesis, transcription, DNA damage repair, cell-cycle control, ubiquitinylation, genetic stability, cell growth and apoptosis. The interacting partners of BRCA1 have given a lot of clue about the functions of this complex protein. In the first project, we used the yeast two-hybrid system to identify novel interacting proteins of BRCA1. We used the 1-500 amino acid region of BRCA1 as bait in library screen and picked up a novel clone (clone 89) showing interaction with BRCA1. Clone 89 contains approximately 2.3 Kb long cDNA sequence. Using the nucleotide blast search, we obtained a full-length cDNA of approximately 5.4 Kb (KIAA0657) that is located on chromosome 2, 2q36.1 region. We have named this new protein BRCA1 Associated Protein 2 (BAP2). Translation of this coding sequence gave a protein that has homology to Titin protein. This protein, which has 1,236 amino acids, contains 9 Immunoglobulin like domains. The homologues of this protein exists in many other organisms but the function is not known. We have confirmed the interaction between BRCA1 and c89 using in vitro GST pull-down assay. We have studied the influence of BAP2 on various functions of BRCA1 like transcription, colony suppression and cell cycle. In the transcription assays, BAP2 activated p21 promoter activity perhaps by using endogenous BRCA1 as simultaneous ectopic expression of truncated BRCA1 (containing aa 1-500) abolished this activity. Further, BAP2 also increased the ability of BRCA1 to activate p21 promoter suggesting that BAP2 may act as a co-activator of BRCA1 functions. Surprisingly, we observed that BAP2 inhibited p53-mediated transcription both in the absence and presence of BRCA1. BAP2 failed to inhibit colony growth by itself as well as in combination with BRCA1. In the cell-cycle study, we found that BAP2 did not have any significant effect on cell cycle profile by itself. However, it drastically augmented the G2/M arrest mediated by BRCA1. Thus we conclude that we have identified a novel interacting protein of BRCA1 that regulates certain functions of BRCA1. Detection of mutations is of central importance in the study of genetic and malignant diseases. Mutation detection helps us in understanding the protein structure, function and expression. More than that, it is also important for pre-symptomatic/antenatal diagnosis, confirmation of the genetic cause of the disease and the mode of inheritance of a disease in a particular family, the prediction of clinical phenotype and the potentiation of diagnostic analysis in the case of families with incomplete pedigrees or with new mutations. Therefore, the importance of direct mutation analysis cannot be understated. The second project deals with screening of mutations in BRCA1 gene in 50 familial breast and/or ovarian cancer families using the technique of Conformation Sensitive Gel Electrophoresis (CSGE). CSGE can be used to detect mismatches in DNA heteroduplexes that contain one strand of wild type and one strand of mutated DNA. In a collaborative study with Kidwai Memorial Hospital for Oncology, Bangalore, we screened 50 families suffering from breast and/or ovarian cancer. We detected 13 mutations in this study out of which 3 are novel and 10 have already been reported earlier (Breast Information Core). All the mutations obtained in our study result in truncation of the BRCA1 protein either because of non-sense mutation or frame-shift mutation. Interestingly, 8 of the mutations detected are 185delAG mutations – the most commonly occurring mutation in Ashkenazi Jewish population. From this study, we conclude that BRCA1 is mutated in 26% of familial breast and/or ovarian cancer cases in India. Genetic testing in individuals with family history of breast, ovarian or both has become very common. It is difficult to interpret the result of genetic screen if a DNA change in the gene does not result in truncation of the protein. Rare missense changes of unknown functional and pathogenic significance are called unclassified variants. It is important to study the functional implications of these unclassified variants in order to determine the risk associated with the presence of such variations. The third project deals with characterization of one such missense variation. In an earlier mutation analysis study for BRCA1 gene in breast cancer samples, we found a novel missense variation resulting in Glu116Lys (E116K) change. In order to determine if this variant is a disease associated missense mutation or a benign sequence alteration; we introduced this variation into full length BRCA1 cDNA and studied its effect on the known functions of BRCA1, namely, transcription, colony suppression and cell cycle. We found that E116K is defective for activating transcription. However, it continued to inhibit growth in colony formation assay and arrest cells in G2/M phase of cell cycle. We conclude that E116K mutation results in loss of transactivation function of BRCA1 but has no effect on colony formation and cell cycle regulation; thus it can be categorized as a novel missense mutation.

Varje år drabbas 4000 personer av malignt melanom i Sverige. Prognosen är god vid tidig upptäckt. Ändå dör ca 20 % av de som drabbas. Ett förbättringsarbete initierades för att utveckla vårdprocesser för dessa patienter. Denna studie har syftat till att utveckla kunskaper om situationsbaserade erfarenheter av vad processen i förbättringsarbetet har inneburit i relation till sjuksköterskors, läkares och medicinska sekreterares egna insatser för projektmålen och en personcentrerad vård. För studien av förbättringsarbetet har Critical Incident Technique (CIT) används som metod. 84 situationer samlades in från projektdeltagare genom korta återkommande intervjuer under projekttiden. Resultatet visar att deltagarna befinner sig i situationer av ambassadörskap, självrannsakan och fristad. Situationer av ambassadörskap är viktiga för projektets riktning framåt men dessa situationer är inte oberoende av situationer av självrannsakan och situationer av ”fristad”. Det är snarare relationen dem emellan som har en viktig dynamisk kraft. Slutsatsen i studien av förbättringsarbetet är att när deltagare i ett förbättringsarbete får möjlighet att ändra på logistik och struktur som bygger på ”stuprörstänk”, professionsorientering och funktion så får det positiva konsekvenser för ett personcentrerat arbetssätt, i arbete med patienter och i arbete med projekt. / Every year 4,000 persons are diagnosed with malignant melanoma in Sweden. The prognosis is good if it is detected early. Nevertheless, about 20% of those who are diagnosed die of the disease. An improvement project was initiated for developing care processes for these patients at a local clinic in central Sweden. This study aimed to describe the knowledge derived from participants in the project regarding their own situated efforts to achieve a person-centered care for these patients. Critical Incident Technique was used as a method. 84 incidents were gathered from the participants through recurrent interviews during the project process. The results show that participants find themselves in situations of being ambassadors for the project where everything flows forward in the project, being in situations of self-examination and doubt about own achievements in the project and finally being in situations of sanctuary as a temporary moral refuge and stress release concerning the project. Situations of ambassadorship are important for the project's direction forward toward the project goal but these situations are not independent of situations of self-examination and situations of sanctuary. The relation between these situations has an important dynamic force that need to be further explored.

Οι φυλετικές ορμόνες επηρεάζουν τη βιολογική συμπεριφορά του μελανώματος. Μετά την ανακάλυψη των οιστρογονικών υποδοχέων (ER) και ιδίως του υποδοχέα βήτα, η άποψη πως τα μελανώματα εκφράζουν χαμηλές συγκεντρώσεις ER αναθεωρήθηκε. Ο σκοπός της παρούσας μελέτης ήταν να διερευνήσει το βαθμό έκφρασης των ER στο πρωτοπαθές μελάνωμα αλλά και στο λεμφαδένα φρουρό αυτού που παριστά ένα σημαντικό σταθμό στη μεταστατική διαδικασία και να εκτιμήσει πιθανή συσχέτιση με την πρόγνωση και τη συνολική επιβίωση. Πραγματοποιήθηκε ανάλυση δεδομένων 60 ασθενών, μέσης ηλικίας 54,4 ± 14,5 ετών, με διάγνωση μελανώματος κατά την περίοδο 2001-2012. Όλοι οι ασθενείς υποβλήθηκαν σε βιοψία λεμφαδένα φρουρού, μετά την αναγνώρισή του με ραδιοϊσοτοπική λεμφαγγειογραφία. Εάν η ιστοπαθολογική εξέταση αναδείκνυε διήθηση του λεμφαδένα από το νεόπλασμα, διενεργείτο συμπληρωματικός λεμφαδενικός καθαρισμός, σε δεύτερο χρόνο. Με ανοσοϊστοχημική μέθοδο εκτιμήθηκε η έκφραση των οιστρογονικών υποδοχέων (ERα και ERβ) σε όλες τις δυνατές περιπτώσεις. Η έκφραση του ERα ήταν εξαιρετικά ασθενής, αναδεικνύοντας τον ERβ σαν τον επικρατούντα οιστρογονικό υποδοχέα, τόσο στον πρωτοπαθή όγκο, όσο και στο λεμφαδένα φρουρό. H εντονότερη ανοσοέκφραση του ERβ καταδείχθηκε σε λεπτά, λιγότερο διηθητικά μελανώματα με αρνητικούς λεμφαδένες φρουρούς. Επιπρόσθετα, η έκφραση του ERβ στον πρωτοπαθή όγκο σχετιζόταν σαφώς με το κυτταρικό μικροπεριβάλλον του, πιθανά επηρεάζοντας τη λεμφογενή διασπορά. Τα επίπεδα έκφρασης του ERβ είναι ελαττωμένα σταθερά σε επιθετικά μελανώματα, μεγάλου πάχους με μεταστατική νόσο στο λεμφαδένα φρουρό. Η αξιολόγησή τους υποδεικνύει έναν πιθανό δείκτη του μεταστατικού δυναμικού των μελανωμάτων και κατ’ επέκταση της πρόγνωσης της νόσου. / Steroid hormones seem to affect the biological behavior of melanoma. Prior to the discovery of estrogen receptor beta (ERb), melanomas were considered to contain low estrogen receptor concentrations. Furthermore, no studies have examined the role of estrogen receptor expression in sentinel lymph nodes (SLNs) of melanomas. The purpose of this study was to investigate the immunoexpression of estrogen receptors in malignant melanomas and SLNs and to examine any possible association with prognosis and overall survival. A retrospective analysis of prospectively collected data was conducted during a 12-year period (2001-2012). Sixty patients with mean age of 54.4 ± 14.5 years old diagnosed with melanomas after excision biopsy of pre-existing melanocytic lesions, were included in the study. All patients underwent wide excision of the primary tumor and SLN identification. Therapeutic lymph node dissection was conducted in cases where the final pathological report was indicative of SLN tumor invasion. Determination of estrogen receptor alpha (ERa) and beta (ERb) status by immunohistochemistry on tumor and nodal paraffin blocks was performed in all feasible cases. ERb but not ERa was the predominant estrogen receptor found in all primary tumors and SLNs examined. The most intense ERb immunostaining was seen in negative SLNs associated with thinner, less invading melanomas. ERb expression in the primary tumor seems to correlate with the cellular microenvironment, possibly altering the process of SLN invasion. ERb expression is down-regulated in aggressive melanomas with sentinel nodal disease, suggesting its possible usefulness as a surrogate marker for metastatic potential and prognosis in malignant melanoma.

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